Electrical signaling is a nearly universal process in cell physiology, initiating and controlling movement, secretion, enzyme activity, and gene expression. Electrical excitability in most vertebrate cells depends on voltage-gated sodium (Na) channels, which open briefly in response to depolarization to allow rapid influx of Na and depolarize the cell to begin the action potential. Impairment of Na channel function causes periodic paralysis, epilepsy, chronic pain, migraine, and cardiac arrhythmia. Elucidation of the molecular basis for electrical signaling is a major challenge for molecular biology and membrane biophysics. Previous studies supported by this research grant led to purification and reconstitution of the Na channel protein and determination of many of its structural and functional properties. In this project period, we have made several advances. We have probed the function of the pore-lining S6 segments in opening of the pore and in slow inactivation. We have shown that regulation by protein phosphorylation by cAMP-dependent protein kinase (PKA) and protein kinase C (PKC) requires specific Na channel/kinase signaling complexes and is dependent on regulation of the intrinsic slow inactivation process of Na channels. In addition, we have analyzed the alterations of activation gating of Na channels by gating modifier toxins, developed a precise molecular model for the function of the voltage sensors in channel activation, and discovered that mutations in the voltage sensor module cause gating pore current-a voltage-gated leak of ions through the mutant voltage sensor into the cell. Remarkably, we found that gating pore current is the pathophysiological mechanism of mutations that cause Hypokalemic Periodic Paralysis (HypoPP) in skeletal muscle. These results focus attention on voltage sensing and activation of Na channels as an important and universal mechanism of cellular excitation and control of cell function, which is often impaired in disease. In the proposed experiments, we will use structure- guided site-directed mutagenesis, ab initio molecular modeling of protein structure, disulfide-crosslinking, and high-resolution biophysical analysis of gating pore currents in order to define the molecular interactions of the gating charges during activation, develop a refined Rosetta sliding helix model for voltage sensor activation, correlate voltage sensor function in Nav1.2/domain IV with fast inactivation and correlate voltage sensor function with slow inactivation and regulation by protein phosphorylation. These studies will reveal the structural basis for voltage-dependent gating by providing a detailed functional map of the Na channel voltage sensors as they initiate channel activation, fast inactivation, slow inactivation, and regulation of slow inactivation by protein phosphorylation. As defects in voltage sensor function are implicated in inherited forms of periodic paralysis, cardiac arrhythmia, epilepsy, and chronic pain, these advances will have direct implications for understanding and treatment of widespread diseases.